泛素基因TaqMan实时荧光定量PCR检测方法的建立及应用  

Development and application of a TaqMan real-time PCR for the detection of ubiquitin gene

作　　者：邓盈盈 谢晶莹[1] 韩玉梅[1,2] 张海霞 李向茸[1,3] 李琼毅[1,2] 魏锁成 冯若飞[1,3] DENG Ying-ying;XIE Jing-ying;HAN Yu-mei;ZHANG Hai-xia;LI Xiang-rong;LI Qiong-yi;WEI Suo-cheng;FENG Ruo-fei(Key Bio-engineering and Technology Laboralory of the Northwest Minzu University,Lanzhou 730030,China;College of Life Science and Engineering,Northwest Minzu University,Lanzhou 730030,China;Gansu Engineering Research Center for Animal Cell,Lanzhou 730030,China)

机构地区：[1]西北民族大学生物工程与技术国家民委重点实验室,甘肃兰州730030 [2]西北民族大学生命科学与工程学院,甘肃兰州730030 [3]甘肃省动物细胞工程技术研究中心,甘肃兰州730030

出　　处：《中国兽医科学》2019年第3期355-362,共8页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31460665;31702234);中央专项研究生科研创新项目(Yxm2018140);教育部"长江学者和创新团队发展计划"项目(IRT_17R88)

摘　　要：为建立一种泛素基因TaqMan实时荧光定量PCR检测方法,以期为泛素基因的动态检测提供有效的试验手段。根据GenBank中公布的泛素基因序列设计1对荧光定量PCR引物及1条探针,建立一种快速检测泛素基因的实时荧光定量PCR方法,并对建立的方法进行条件优化与评价。结果显示,建立的实时荧光定量PCR方法的线性关系好,以质粒标准品构建的标准曲线的相关系数r^2为0.999;灵敏性、特异性及重复性均较高;应用建立的方法对多种种属来源细胞进行检测,能检测出不同类型细胞中泛素的含量。结果表明,成功建立了一种快速、准确检测泛素TaqMan实时荧光定量PCR方法。To develop a real-time TaqMan reverse transcription-PCR(RT-PCR)assay for effective detection of ubiquitin gene,based on the ubiquitin gene sequence published in GenBank,a pair of PCR primers and a probe were designed.Then a real-time TaqMan RT-PCR assay for accurate quantification of ubiquitin gene was developed and optimized.Results showed that the real-time TaqMan RT-PCR assay had a good linear relationship.The standard curve correlation coefficient r^2 was 0.999.It also had a high sensitivity and reproducibility as well as specificity.The method was used to detect the contents of ubiquitin in different types of cells.In conclusion,a rapid and accurate real-time Taq Man reverse transcription-PCR for quantification of ubiquitin gene was successfully developed.

关 键 词：泛素 实时荧光定量PCR TAQMAN探针 

分 类 号：S852.21[农业科学—基础兽医学]